Research On Enzymic Basis Of Acarbose Analogues Formation By Actinoplanes Sp.Zjb03852 And Their Control During Fermentation

Acarbose,industrially produced by Actinoplanes sp.,is an a-glucosidase inhibitor and widely used in the treatment of non-insulin dependent diabetes mellitus.Depending on its strong inhibitory effect towards intestinal sucrase,maltase as well as glucamylase,acarbose can reduce blood glucose level.It has been widely used because of its excellent pharmacokinetic and high safety.Besides of acarbse,some analogues containing acarviosyl can be produced in the fermentation of Actinoplan.es sp.These analogues can cause difficulties in the downstream separation of acarbose and reduce the inhibition effect on a-glucosidase.In order to analyze the factors involved in the formation and inhibition of these analogues and ultimately reduce impurities content in the fermentation broth,the related glucosyltransferases were purified and their characteristics were studied.Firstly,impurity C and impurity F,produced by catalyzing acarbose and oligosaccharides using intracellular or extracellular extract of Actinoplanes sp.ZJB03852,were determined by LC-MS analysis.The conditions of intracellular and extracellular extract glucosyltransferase activity were studied.Adding trehalose could promote the intracellular glucosyltransferase activity,while maltose inhibit.Transglycosylation activity in the extract did not change with the prolonging of Actinoplanes sp.ZJB03852 fermentation.When the maltose syrup in the initial culture medium was replaced by the mixture of same amount of maltose and trehalose,transglycosylation activity was promoted.But starch did not.Impurity C was intramolecularly converted from acarbose by the catalysis of the intracellular extract,while impurity F was formed by transferring acarvoisyl moiety in acarbose to maltotriose catalyzed by extracellular extract.As for the extracellular extract,trans-acarvoisylaton activity decreased as the fermentation going on.Maltose syrup in the initial culture medium replaced by starch could enhance trans-acarvoisylaton activity,but could not facilitate the production of acarbose in fermentation broth.Actinoplanes sp.ZJB03852 intracellular glucosyltransferase was purified by the steps including ultrasonic cell disruption,ammonium sulfate fraction precipitation,Micro-prep DEAE anion-exchange chromatography,Micro-prep t-Butyl hydrophobic interaction chromatography and UNO-Q anion-exchange chromatography.The specific activity of 15.6 U·mg^-1 of the purified protein was 48.8-fold from the crude extracts with a yield of 6.8%.The molecular weight of glucosyltransferase was about 51 kDa by analysis through SDS-PAGE.The characterization of the enzyme was studied.The optimum temperature and pH of glucosyltransferase was 25℃ and 7.5,respectively.The enzyme appeared to be stable below 40℃ and pH 6.0-7.5,respectively.The enzyme activity was enhanced in the presence of Li+,while partially inhibited by Zn²⁺,Fe²⁺,Fe³⁺and Al³⁺.One of validamycin derivatives,validamine,could also inhibit the enzyme activity.Kinetic analysis showed that the Km and Vmax of glucosyltransferase were 6.89 g·L^-1 and 46.72 g·L^-1·h^-1,respectively.Validamine,valienamine and valiolamine were competitive inhibitor of the glucosyltransferase.Validamine was one of the most effective inhibitor.At last,Actinoplanes sp.ZJB03852 acarvoisyl transferase was purified by ammonium sulfate fraction precipitation,Micro-prep High Q anion-exchange chromatography,Micro-prept-Butyl hydrophobic interaction chromatography and UNO-Q anion-exchange chromatography.The specific activity of 8.3 U·mg^-1 of the purified protein was 16.6-fold from the crude enzyme with a yield of 17.8%.The molecular weight of acarviosyl transferase was about 75 kDa by analysis through SDS-PAGE.The characterization of the enzyme was studied.The optimum temperature and pH of acarviosyl transferase was 30℃ and between 6.5～8.0,respectively.The enzyme appeared to be stable below 20℃ and pH 6.0-8.0,respectively.The enzyme activity was enhanced in the presence of K+,while partially inhibited by Co²⁺,Mg²⁺,Cu²⁺,Fe³⁺and Al³⁺.